The present application extends a successful multifaceted investigation of host resistance to viral infection. The strengths of our approach include: 1) an unbiased component based on mutagenesis combined with a hypothesis-driven component; 2) the study of distantly related organisms (mice and Drosophila) to appreciate which elements of defense are conserved; 3) the embrace of new and powerful methods to support our efforts. In our work to date, we have collectively identified new sensors (e.g., LGP2; DICER-2) necessary for activation of antiviral defenses, and delineated pathways of response to viruses, both at a biochemical level and in terms of communication between cells. We have identified previously unknown molecular participants in host defense. Among these are channel proteins (SLC15A4; KCNJ8/SUR2), transcription factors (IKB;AKIRIN2), proteins concerned with membrane trafficking or organellar mobility (AP3B1; STING; TR1M56; ATG9A; UNC93B), cell stress (SLFN2), post-translational modification (TRIM56; TR1M23), and endosome physiology (SLC15A4). Some of these proteins are members of extended families and may open the way to broad new models of host defense. Others highlight the importance of intermediary steps in host defense (e.g., the movement of molecules within cells) in a way that has not been considered before. Each participating group (Dallas, Osaka, and Strasbourg) has its special talents, and these have been combined to take us beyond genetics per se, incorporating new technologies that will accelerate the discovery of essential elements of the host defense apparatus. We recognize that it is not enough to possess a list of parts to understand how a machine operates. As new proteins are shown to be essential for particular aspects of host defense, we will establish how they interact with one another and/or other proteins to support resistance; how they catalyze particular reactions within cells, and how they drive or suppress the expression of genes in what we see as a highly dynamic process. We view the continuation of this POl as an opportunity to build upon an approach with established productivity: one that has generated new molecules, concepts, and reagents for use by the scientific community as a whole. The POl has been, and will continue to be, highly collaborative in the exchange of methods, genetic materials, and most importantly, ideas, ultimately derived from genetics. PUBLIC HEALTH RELEVANCE: Historically, viruses have been responsible for more death among humans than any other single cause, and still constitute a serious threat to mankind. We will use strong genetic techniques to study both host resistance mechanisms (innate immunity to viral infection), and the essential requirements that viruses have in order to cause infection. Basic scientific discovery in these areas will pave the way to the design of effective anti-viral drugs and vaccines, and has the highest relevance to human health. Project 1: Forward genetic analysis of Mammalian Resistance to Viral Infection Project 1 Leader (PL): Bruce Beutler DESCRIPTION (provided by applicant): We will extend our successful forward genetic analysis of susceptibility to mouse cytomegalovirus (MCMV) infection, and institute a new screen for cell-autonomous resistance to Rift Valley Fever Virus (RVFV). These studies will build upon technological advances that permit us to find mutations faster than ever before, using massively parallel sequencing, both by itself and in combination with inducible pluripotent stem cell (IPS) technology. We have found that it is possible to screen mutagenized MEFs from reprogrammable C57BL/6J mice for resistance to RVFV ex vivo, to identify resistant clones, to use these clones to regenerate live mice, and at the same time, detect most of the mutations in these cells by sequencing genomic DNA. We have determined, based on in silico simulations, that compound heterozygous null alleles at almost all loci will result from mutagenesis on the scale we contemplate. As such, we hope to enrich our understanding of what it takes to combat a model herpesvirus infection with strong relevance to a human disease (HCMV infection), and also, to determine the critical host proteins necessary for a Category A pathogen (RVFV) to proliferate in mammalian cells. This work, pursued in depth, will inform us of molecular targets for the treatment of bunyavirus infections in general. In a third, circumscribed specific aim, we will analyze a new mutation, identified in screening that abolishes the responses of plasmacytoid dendritic cells (PDCs) to nucleic acids. This mutation, called feeble, affects a channel protein that acts in conjunction with the adaptor protein 3 (AP3) complex to condition endosomes in PDCs (but not other cells), making them competent to signal via TLRs. Based on our studies to date, we infer the existence of an ARF1-->AP3 -->SIc45a-->TLR7/9 pathway essential for the support of type I interferon gene induction in PDCs. The further elucidation of this pathway will deepen understanding of PDC function, and the role of PDC-derived interferon both in infection and in pathological settings such as systemic lupus erythematosus.